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1.
J Biosci Bioeng ; 131(1): 8-12, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33023861

RESUMO

Sphingomyelinase C (SMC) hydrolyzes sphingomyelin to ceramide and phosphocholine. Prokaryotic SMCs share sequence homology with mammalian SMCs that have enzymatic pH optima at neutral pH. SMC from the nonpathogenic prokaryote Streptomyces griseocarneus shows notable enzymatic features such as higher optimum pH and thermostability than other prokaryotic SMCs. Determination of the three-dimensional structure of S. griseocarneus-SMC (Sg-SMC) and comparison with other SMC structures represents a promising strategy to elucidate the unique enzymatic features of Sg-SMC on a structural basis. Therefore, we determined the crystal structure of Sg-SMC at 2.0 Å resolution by X-ray crystallography. Comparison of the Sg-SMC structure with three other structurally known SMCs from Listeria ivanovii, Bacillus cereus, and Staphylococcus aureus indicated that Sg-SMC is more diverse in sequence and that structural differences in the main chain between these SMCs are primarily located on the molecular surface distant from the active site. Comparison of the surface area of the four SMCs revealed that Sg-SMC has the most compact structure, which may contribute to the enhanced thermostability of Sg-SMC. Regarding the hydrogen bond network in the active site of Sg-SMC, a basic amino acid, Arg278, is involved, whereas the corresponding residue in other SMCs (Ser or Asn) does not form hydrogen bonds with metal-coordinating water molecules. Hydrogen bond formation between Arg278 and a Mg2+ ion-coordinating water molecule may be responsible for the higher optimal pH of Sg-SMC compared to that of other SMCs.


Assuntos
Esfingomielina Fosfodiesterase/química , Esfingomielina Fosfodiesterase/metabolismo , Streptomyces/enzimologia , Temperatura , Animais , Domínio Catalítico , Cristalografia por Raios X , Estabilidade Enzimática , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio
2.
Food Chem ; 340: 127903, 2021 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-32889205

RESUMO

Microbial transglutaminase (mTG) catalyses the formation of protein crosslinks, deamidating glutamine in a side-reaction. Gluten deamidation by human tissue transglutaminase is critical to activate celiac disease pathogenesis making the addition of mTG to wheat-based products controversial. The ability of mTG (0-2000 U.kg-1) to alter gluten's structure, digestibility and the deamidation state of six immunogenic gluten peptides within bread was investigated. Gluten's structure was altered when mTG exceeded 100 U.kg-1, determined by confocal microscopy, extractability and free sulfhydryl assays. The effect of mTG on six immunogenic peptides was investigated by in vitro digestion (INFOGEST) and mass spectrometry. The addition of mTG to bread (0-2000 U.kg-1) did not alter the deamidation state or digestibility of the immunogenic peptides investigated. Overall, this investigation indicated that the addition of mTG to bread does not create activated gluten peptides. This analysis provides evidence for risk assessments of mTG as a food processing aid.


Assuntos
Pão , Glutens/química , Glutens/farmacocinética , Transglutaminases/metabolismo , Pão/análise , Doença Celíaca , Digestão , Glutens/imunologia , Humanos , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/imunologia , Proteólise , Streptomyces/enzimologia , Transglutaminases/química , Triticum/química
3.
Proc Natl Acad Sci U S A ; 117(40): 24794-24801, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-32958639

RESUMO

The structure of lincomycin A consists of the unusual eight-carbon thiosugar core methyllincosamide (MTL) decorated with a pendent N-methylprolinyl moiety. Previous studies on MTL biosynthesis have suggested GDP-ᴅ-erythro-α-ᴅ-gluco-octose and GDP-ᴅ-α-ᴅ-lincosamide as key intermediates in the pathway. However, the enzyme-catalyzed reactions resulting in the conversion of GDP-ᴅ-erythro-α-ᴅ-gluco-octose to GDP-ᴅ-α-ᴅ-lincosamide have not yet been elucidated. Herein, a biosynthetic subpathway involving the activities of four enzymes-LmbM, LmbL, CcbZ, and CcbS (the LmbZ and LmbS equivalents in the closely related celesticetin pathway)-is reported. These enzymes catalyze the previously unknown biosynthetic steps including 6-epimerization, 6,8-dehydration, 4-epimerization, and 6-transamination that convert GDP-ᴅ-erythro-α-ᴅ-gluco-octose to GDP-ᴅ-α-ᴅ-lincosamide. Identification of these reactions completes the description of the entire lincomycin biosynthetic pathway. This work is significant since it not only resolves the missing link in octose core assembly of a thiosugar-containing natural product but also showcases the sophistication in catalytic logic of enzymes involved in carbohydrate transformations.


Assuntos
Lincomicina/biossíntese , Streptomyces/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Vias Biossintéticas , Lincomicina/química , Lincosamidas/química , Lincosamidas/metabolismo , Streptomyces/química , Streptomyces/enzimologia , Streptomyces/genética
4.
Nat Commun ; 11(1): 4022, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32782248

RESUMO

One major bottleneck in natural product drug development is derivatization, which is pivotal for fine tuning lead compounds. A promising solution is modifying the biosynthetic machineries of middle molecules such as macrolides. Although intense studies have established various methodologies for protein engineering of type I modular polyketide synthase(s) (PKSs), the accurate targeting of desired regions in the PKS gene is still challenging due to the high sequence similarity between its modules. Here, we report an innovative technique that adapts in vitro Cas9 reaction and Gibson assembly to edit a target region of the type I modular PKS gene. Proof-of-concept experiments using rapamycin PKS as a template show that heterologous expression of edited biosynthetic gene clusters produced almost all the desired derivatives. Our results are consistent with the promiscuity of modular PKS and thus, our technique will provide a platform to generate rationally designed natural product derivatives for future drug development.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Policetídeo Sintases/genética , Produtos Biológicos/química , Produtos Biológicos/metabolismo , Estrutura Molecular , Família Multigênica/genética , Policetídeo Sintases/metabolismo , Sirolimo/química , Sirolimo/metabolismo , Estereoisomerismo , Streptomyces/enzimologia , Streptomyces/genética , Streptomyces/metabolismo
5.
Arch Biochem Biophys ; 692: 108544, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-32822639

RESUMO

Rapamycin is a clinically important macrolide agent with immunosuppressant and antiproliferative properties, produced by the actinobacterium, Streptomyces rapamycinicus. Two cytochrome P450 enzymes are involved in the biosynthesis of rapamycin. CYP107G1 and CYP122A2 catalyze the oxidation reactions of C27 and C9 of pre-rapamycin, respectively. To understand the structural and biochemical features of P450 enzymes in rapamycin biosynthesis, the CYP107G1 and CYP122A2 genes were cloned, their recombinant proteins were expressed in Escherichia coli, and the purified enzymes were characterized. Both enzymes displayed low spin states in the absolute spectra of ferric forms, and the titrations with rapamycin induced type I spectral changes with Kd values of 4.4 ± 0.4 and 3.0 ± 0.3 µM for CYP107G1 and CYP122A2, respectively. The X-ray crystal structures of CYP107G1 and its co-crystal complex with everolimus, a clinical rapamycin derivative, were determined at resolutions of 2.9 and 3.0 Å, respectively. The overall structure of CYP107G1 adopts the canonical scaffold of cytochrome P450 and possesses large substrate pocket. The distal face of the heme group is exposed to solvents to accommodate macrolide access. When the structure of the everolimus-bound CYP107G1 complex (CYP107G1-Eve) was compared to that of the ligand-free CYP107G1 form, no significant conformational change was observed. Hence, CYP107G1 has a relatively rigid structure with versatile loops to accommodate a bulky substrate. The everolimus molecule is bound to the substrate-binding pocket in the shape of a squeezed donut, and its elongated structure is bound perpendicular to a planar heme plane and I-helix.


Assuntos
Proteínas de Bactérias/química , Sistema Enzimático do Citocromo P-450/química , Streptomyces/enzimologia , Proteínas de Bactérias/genética , Cristalografia por Raios X , Sistema Enzimático do Citocromo P-450/genética , Domínios Proteicos , Estrutura Secundária de Proteína , Proteínas Recombinantes , Sirolimo/metabolismo , Streptomyces/genética
6.
J Med Chem ; 63(16): 8867-8875, 2020 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-32787146

RESUMO

Rare neglected diseases may be neglected but are hardly rare, affecting hundreds of millions of people around the world. Here, we present a hit identification approach using AtomNet, the world's first deep convolutional neural network for structure-based drug discovery, to identify inhibitors targeting aspartate N-acetyltransferase (ANAT), a promising target for the treatment of patients suffering from Canavan disease. Despite the lack of a protein structure or high sequence identity homologous templates, the approach successfully identified five low-micromolar inhibitors with drug-like properties.


Assuntos
Acetiltransferases/antagonistas & inibidores , Aprendizado Profundo , Descoberta de Drogas/métodos , Inibidores Enzimáticos/química , Humanos , Estrutura Molecular , Streptomyces/enzimologia
7.
Arch Biochem Biophys ; 691: 108489, 2020 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-32697946

RESUMO

2-Phosphinomethylmalate synthase (PMMS) from Streptomyces hygroscopicus catalyzes the first step in the biosynthesis of the herbicide bialophos using 3-phosphinopyruvic acid and acetyl coenzyme A as substrates to form 2-phosphinomethylmalic acid and coenzyme A. PMMS belongs to the Claisen condensation-like (CC-like) subgroup of the DRE-TIM metallolyase superfamily, which uses conserved active site architecture to catalyze a functionally-diverse set of reactions. Analysis of a sequence similarity network for the CC-like subgroup identified PMMS and the related R-citrate synthase in an early-diverging cluster suggesting that this group of sequences are more distinct in relation to other Claisen-condensation subgroup members. To better understand the structure/function landscape of the CC-like subgroup PMMS was recombinantly expressed in Escherichia coli, purified, and characterized with respect to its enzymatic properties. Using oxaloacetate as a substrate analog, the recombinantly-produced enzyme exhibited improved Michaelis constants relative to the previously reported natively-produced enzyme. Results from pH rate profiles and kinetic isotope effects were consistent with results from other members of the CC-like subgroup supporting acid-base chemistry and hydrolysis of the direct Claisen-condensation product as the rate-determining step. Results of site-directed mutagenesis experiments indicate that PMMS uses an active-site architecture similar to homocitrate synthase to select for a dicarboxylic acid substrate.


Assuntos
Proteínas de Bactérias/química , Carbono-Carbono Liases/química , Streptomyces/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Carbono-Carbono Liases/genética , Carbono-Carbono Liases/isolamento & purificação , Catálise , Domínio Catalítico/genética , Escherichia coli/genética , Cinética , Mutagênese Sítio-Dirigida , Mutação , Ácido Oxaloacético/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação
8.
Nat Chem Biol ; 16(9): 1013-1018, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32601484

RESUMO

D-amino acids endow peptides with diverse, desirable properties, but the post-translational and site-specific epimerization of L-amino acids into their D-counterparts is rare and chemically challenging. Bottromycins are ribosomally synthesized and post-translationally modified peptides that have overcome this challenge and feature a D-aspartate (D-Asp), which was proposed to arise spontaneously during biosynthesis. We have identified the highly unusual α/ß-hydrolase (ABH) fold enzyme BotH as a peptide epimerase responsible for the post-translational epimerization of L-Asp to D-Asp during bottromycin biosynthesis. The biochemical characterization of BotH combined with the structures of BotH and the BotH-substrate complex allowed us to propose a mechanism for this reaction. Bioinformatic analyses of BotH homologs show that similar ABH enzymes are found in diverse biosynthetic gene clusters. This places BotH as the founding member of a group of atypical ABH enzymes that may be able to epimerize non-Asp stereocenters across different families of secondary metabolites.


Assuntos
Racemases e Epimerases/química , Racemases e Epimerases/metabolismo , Ácido Aspártico/química , Ácido Aspártico/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Evolução Molecular , Modelos Moleculares , Família Multigênica , Peptídeos Cíclicos/metabolismo , Conformação Proteica , Dobramento de Proteína , Racemases e Epimerases/genética , Streptomyces/enzimologia , Streptomyces/genética , Especificidade por Substrato
9.
Inorg Chem ; 59(14): 9627-9637, 2020 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-32644783

RESUMO

The b-type cytochrome LcpK30 is a latex clearing protein (Lcp), which acts as an endotype dioxygenase to catalyze the extracellular cleavage of the chemically inert aliphatic polymer poly(cis-1,4-isoprene), producing oligo-isoprenoids with different terminal carbonyl groups (aldehyde and ketone, -CH2-CHO and -CH2-COCH3). On the basis of the fact that the muteins of E148A, E148Q, and E148H have substantially reduced reactivity, and the E148-initiated reaction mechanism has been previously proposed, in which a cyclic dioxetane intermediate or an epoxide intermediate may be involved, however, open questions still remain. In this paper, on the basis of the crystal structure of LcpK30, the enzyme-substrate reactant model was constructed, and the cleavage mechanism of the central double bond of poly(cis-1,4-isoprene) was elucidated by performing quantum mechanics/molecular mechanics calculations. Our calculation results revealed that the oxidative cleavage reaction is triggered by the addition of the heme-bound dioxygen to the double bond of the polymer, and E148 does not act as the catalytic base to extract the allylic proton to assist the reaction as previously suggested. Of the two considered pathways, the pathway that involves the dioxetane intermediate was calculated to be more favorable. During the catalysis, the distal oxygen first adds to the double bond of the substrate to form a radical intermediate, and then the Fe-O1 (proximal oxygen) bond cleaves to generate the dioxetane intermediate, which can easily collapse affording the final ketone and aldehyde products. In general, the cleavage mechanism of double C-C bond catalyzed by LcpK30 is similar to those of indoleamine 2,3-dioxygenase, tryptophan 2,3-dioxygenase, and the nonheme stilbene cleavage oxygenase NOV1 that all depend on the iron-bound dioxygen to initiate the cleavage reaction.


Assuntos
Proteínas de Bactérias/química , Dioxigenases/química , Hemiterpenos/química , Látex/química , Proteínas de Bactérias/genética , Domínio Catalítico , Dioxigenases/genética , Heme/química , Modelos Químicos , Simulação de Acoplamento Molecular , Mutação , Oxigênio/química , Teoria Quântica , Streptomyces/enzimologia
10.
Nucleic Acids Res ; 48(15): 8755-8766, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32621606

RESUMO

The sulfur atom of phosphorothioated DNA (PT-DNA) is coordinated by a surface cavity in the conserved sulfur-binding domain (SBD) of type IV restriction enzymes. However, some SBDs cannot recognize the sulfur atom in some sequence contexts. To illustrate the structural determinants for sequence specificity, we resolved the structure of SBDSpr, from endonuclease SprMcrA, in complex with DNA of GPSGCC, GPSATC and GPSAAC contexts. Structural and computational analyses explained why it binds the above PT-DNAs with an affinity in a decreasing order. The structural analysis of SBDSpr-GPSGCC and SBDSco-GPSGCC, the latter only recognizes DNA of GPSGCC, revealed that a positively charged loop above the sulfur-coordination cavity electrostatically interacts with the neighboring DNA phosphate linkage. The structural analysis indicated that the DNA-protein hydrogen bonding pattern and weak non-bonded interaction played important roles in sequence specificity of SBD protein. Exchanges of the positively-charged amino acid residues with the negatively-charged residues in the loop would enable SBDSco to extend recognization for more PT-DNA sequences, implying that type IV endonucleases can be engineered to recognize PT-DNA in novel target sequences.


Assuntos
Enzimas de Restrição do DNA/genética , Proteínas de Ligação a DNA/genética , DNA/genética , Enxofre/química , Sequência de Aminoácidos/genética , Cristalografia por Raios X , DNA/química , Enzimas de Restrição do DNA/química , Proteínas de Ligação a DNA/química , Escherichia coli/genética , Ligação de Hidrogênio , Ligação Proteica/genética , Domínios Proteicos/genética , Streptomyces/enzimologia
11.
J Med Microbiol ; 69(8): 1040-1048, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32692643

RESUMO

Given the increased reporting of multi-resistant bacteria and the shortage of newly approved medicines, researchers have been looking towards extreme and unusual environments as a new source of antibiotics. Streptomyces currently provides many of the world's clinical antibiotics, so it comes as no surprise that these bacteria have recently been isolated from traditional medicine. Given the wide array of traditional medicines, it is hoped that these discoveries can provide the much sought after core structure diversity that will be required of a new generation of antibiotics. This review discusses the contribution of Streptomyces to antibiotics and the potential of newly discovered species in traditional medicine. We also explore how knowledge of traditional medicines can aid current initiatives in sourcing new and chemically diverse antibiotics.


Assuntos
Antibacterianos/isolamento & purificação , Descoberta de Drogas/tendências , Microbiologia do Solo , Streptomyces/metabolismo , Animais , Antibacterianos/biossíntese , Cavernas/química , Invertebrados/química , Medicina Tradicional , Peptídeo Sintases/metabolismo , Plantas Medicinais/química , Policetídeo Sintases/metabolismo , Poríferos/química , Streptomyces/química , Streptomyces/enzimologia
12.
Sci Rep ; 10(1): 12021, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32694623

RESUMO

Streptomycetes are filamentous bacteria famous for their ability to produce a vast majority of clinically important secondary metabolites. Both complex morphogenesis and onset of antibiotic biosynthesis are tightly linked in streptomycetes and require series of specific signals for initiation. Cyclic dimeric 3'-5' guanosine monophosphate, c-di-GMP, one of the well-known bacterial second messengers, has been recently shown to govern morphogenesis and natural product synthesis in Streptomyces by altering the activity of the pleiotropic regulator BldD. Here we report a role of the heme-binding diguanylate cyclase SSFG_02181 from Streptomyces ghanaensis in the regulation of the peptidoglycan glycosyltransferase inhibitor moenomycin A biosynthesis. Deletion of ssfg_02181 reduced the moenomycin A accumulation and led to a precocious sporulation, while the overexpression of the gene blocked sporogenesis and remarkably improved antibiotic titer. We also demonstrate that BldD negatively controls the expression of ssfg_02181, which stems from direct binding of BldD to the ssfg_02181 promoter. Notably, the heterologous expression of ssfg_02181 in model Streptomyces spp. arrested morphological progression at aerial mycelium level and strongly altered the production of secondary metabolites. Altogether, our work underscores the significance of c-di-GMP-mediated signaling in natural product biosynthesis and pointed to extensively applicable approach to increase antibiotic production levels in streptomycetes.


Assuntos
Antibacterianos/biossíntese , Bambermicinas/biossíntese , GMP Cíclico/análogos & derivados , Proteínas de Escherichia coli/metabolismo , Engenharia Metabólica/métodos , Fósforo-Oxigênio Liases/metabolismo , Streptomyces/enzimologia , Streptomyces/crescimento & desenvolvimento , Proteínas de Bactérias/metabolismo , GMP Cíclico/genética , GMP Cíclico/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Ligantes de Grupo Heme/genética , Proteínas Ligantes de Grupo Heme/metabolismo , Morfogênese/genética , Fósforo-Oxigênio Liases/genética , Regiões Promotoras Genéticas , Sistemas do Segundo Mensageiro/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
13.
Sheng Wu Gong Cheng Xue Bao ; 36(5): 932-941, 2020 May 25.
Artigo em Chinês | MEDLINE | ID: mdl-32567276

RESUMO

Endo-ß-N-acetylglucosaminidase is used widely in the glycobiology studies and industries. In this study, a new endo-ß-N-acetylglucosaminidase, designated as Endo SA, was cloned from Streptomyces alfalfae ACCC 40021 and expressed in Escherichia coli BL21 (DE3). The purified recombinant Endo SA exhibited the maximum activity at 35 ºC and pH 6.0, good thermo/pH stability and high specific activity (1.0×106 U/mg). It displayed deglycosylation activity towards different protein substrates. These good properties make EndoSA a potential tool enzyme and industrial biocatalyst.


Assuntos
Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidase , Streptomyces , Clonagem Molecular , Estabilidade Enzimática , Escherichia coli/genética , Expressão Gênica , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidase/genética , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidase/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Streptomyces/enzimologia , Streptomyces/genética
14.
J Ind Microbiol Biotechnol ; 47(6-7): 537-542, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32588231

RESUMO

Alizarin has been reported to have an antigenotoxic activity along with an inhibitory effect on the tumor cell growth of human colon carcinoma cells. Alizarin was biotransformed into an O-methoxide derivative using O-methyltransferase from Streptomyces avermitilis MA4680 (SaOMT2) to enhance its bioefficacy. The biotransformed product was extracted, purified, and characterized using various chromatographic and spectroscopic analyses, and confirmed to be an alizarin 2-O-methoxide. The antiproliferative activity of the compound against gastric cancer cells (AGS), uterine cervical cancer (Hela), liver cancer (HepG2), and normal cell lines was investigated. Alizarin 2-O-methoxide showed an inhibitory effect on all three cancer-cell lines at very low concentrations, from 0.078 µM, with no cytotoxicity against 267B1 (human prostate epithelial) and MRC-5 (normal human fetal lung fibroblast).


Assuntos
Antraquinonas/metabolismo , Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Neoplasias/patologia , Streptomyces/enzimologia , Biotransformação , Linhagem Celular Tumoral , Escherichia coli , Células HeLa , Células Hep G2 , Humanos , Microbiologia Industrial , Concentração Inibidora 50 , Espectroscopia de Ressonância Magnética , Neoplasias/tratamento farmacológico
15.
Arch Microbiol ; 202(7): 1597-1615, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32451592

RESUMO

Extracellular enzymes produced from Streptomyces have the potential to replace toxic chemicals that are being used in various industries. The endorsement of this replacement has not received a better platform in developing countries. In this review, we have discussed the impact of chemicals and conventional practices on environmental health, and the role of extracellular enzymes to replace these practices. Burning of fossil fuels and agriculture residue is a global issue, but the production of biofuel using extracellular enzymes may be the single key to solve all these issues. We have discussed the replacement of hazardous chemicals with the use of xylanase, cellulase, and pectinase in food industries. In paper industries, delignification was done by the chemical treatment, but xylanase and laccase have the efficient potential to remove the lignin from pulp. In textile industries, the conventional method includes the chemicals which affect the nervous system and other organs. The use of xylanase, cellulase, and pectinase in different processes can give a safe and environment-friendly option to textile industries. Hazardous chemical pesticides can be replaced by the use of chitinase as an insecticide and fungicide in agricultural practices.


Assuntos
Proteínas de Bactérias/metabolismo , Enzimas/metabolismo , Microbiologia Industrial/tendências , Streptomyces/enzimologia , Agricultura , Biocombustíveis , Lignina/metabolismo
16.
Enzyme Microb Technol ; 137: 109519, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32423668

RESUMO

Inulin is a widespread polysaccharide, and it can be bio-transferred to α-d-fructofuranose-ß-d-fructofuranose 1,2':2,1'-dianhydride (DFA I), a potential low-calorie sweetener, via inulin fructotransferase (IFTase, EC 4.2.2.17). In this study, a novel DFA I-forming IFTase was identified and characterized from Streptomyces peucetius subsp. caesius ATCC 27952 (SpIFTase). The specific activity of SpIFTase was determined to be 420.84 ± 6.21 U mg-1 at pH 6.5 and 45 °C. The enzyme exhibited a prominent thermostability with a half-life of 70 min at 70 °C and a structural melting temperature (Tm) of 75.48 °C. The values of Km and kcat/Km of SpIFTase against inulin substrate were 3.08 mM and 199.09 mM-1 s-1, respectively. Furthermore, the catalytic residues were investigated by site-directed mutagenesis with an alanine scanning method. Interestingly, the critical catalytic residues of SpIFTase were speculated to be residues D162 and E231, which were firstly non-conserved with those of previously reported IFTases. This work proposes a potential for industrial DFA I production and new insights into the catalytic mechanism of DFA I-forming IFTases.


Assuntos
Hexosiltransferases/genética , Hexosiltransferases/metabolismo , Streptomyces/enzimologia , Edulcorantes/metabolismo , Temperatura , Biocatálise , Biotransformação , Clonagem Molecular , Estabilidade Enzimática , Genes Bacterianos , Concentração de Íons de Hidrogênio , Mutagênese Sítio-Dirigida , Streptomyces/genética
17.
Enzyme Microb Technol ; 137: 109536, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32423673

RESUMO

N-acylated amino acids are widely used as surfactants and/or actives in cosmetics and household formulations. Their industrial production is based on the use of the Schotten-Baumann chemical and unselective reaction. Faced to the growing demand for greener production processes, selective enzymatic synthesis in more environment-friendly conditions starts to be considered as a potential alternative. This study concerns the use of the aminoacylases from Streptomyces ambofaciens to selectively catalyse aminoacid acylation reaction by fatty acids in aqueous medium. The results demonstrated that, when using undecylenoic acid as acyl donor, these aminoacylases properly catalyse the acylation of 14 of the 20 proteogenic l-amino acids tested on their α amino group with a great variability depending on the nature of the amino acid (polar or not, positively/negatively charged, aromatic or not…). More precisely, the following 9 amino acids were shown to be preferentially acylated by S. ambofaciens aminoacylases as follows: lysine > arginine > leucine > methionine > phenylalanine > valine > cysteine > isoleucine > threonine. Different fatty acids were used as acyl donors and, in most cases, the fatty acid length influenced the conversion yield. The kinetic study of α-lauroy-lysine synthesis showed a positive influence of lysine concentration with Vmax and Km of 3.7 mM/h and 76 mM, respectively. Besides, the lauric acid had an inhibitory effect on the reaction with Ki of 70 mM. The addition of cobalt to the reaction medium led to a more than six-fold increase of the reaction rate. These results, achieved with the aminoacylases from S. ambofaciens represent an improved enzyme-based N-acylated amino acids production in order to provide an alternative way to the Schotten-Baumann chemical reaction currently used in the industry.


Assuntos
Amidoidrolases/metabolismo , Aminoácidos/metabolismo , Biocatálise , Streptomyces/enzimologia , Acilação , Cobalto/metabolismo , Cinética
18.
Nat Chem Biol ; 16(7): 776-782, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32367018

RESUMO

In type II polyketide synthases (PKSs), the ketosynthase-chain length factor (KS-CLF) complex catalyzes polyketide chain elongation with the acyl carrier protein (ACP). Highly reducing type II PKSs, represented by IgaPKS, produce polyene structures instead of the well-known aromatic skeletons. Here, we report the crystal structures of the Iga11-Iga12 (KS-CLF) heterodimer and the covalently cross-linked Iga10=Iga11-Iga12 (ACP=KS-CLF) tripartite complex. The latter structure revealed the molecular basis of the interaction between Iga10 and Iga11-Iga12, which differs from that between the ACP and KS of Escherichia coli fatty acid synthase. Furthermore, the reaction pocket structure and site-directed mutagenesis revealed that the negative charge of Asp 113 of Iga11 prevents further condensation using a ß-ketoacyl product as a substrate, which distinguishes IgaPKS from typical type II PKSs. This work will facilitate the future rational design of PKSs.


Assuntos
Proteína de Transporte de Acila/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Ácido Graxo Sintases/química , Policetídeo Sintases/química , Policetídeos/química , Proteína de Transporte de Acila/genética , Proteína de Transporte de Acila/metabolismo , Biocatálise , Domínio Catalítico , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Ácido Graxo Sintases/genética , Ácido Graxo Sintases/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Modelos Moleculares , Mutagênese Sítio-Dirigida , Policetídeo Sintases/genética , Policetídeo Sintases/metabolismo , Policetídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Streptomyces/enzimologia , Streptomyces/genética , Especificidade por Substrato
19.
Sci Rep ; 10(1): 7813, 2020 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-32385385

RESUMO

The conversion of lignocellulosic biomass into bioethanol or biochemical products requires a crucial pretreatment process to breakdown the recalcitrant lignin structure. This research focuses on the isolation and characterization of a lignin-degrading bacterial strain from a decaying oil palm empty fruit bunch (OPEFB). The isolated strain, identified as Streptomyces sp. S6, grew in a minimal medium with Kraft lignin (KL) as the sole carbon source. Several known ligninolytic enzyme assays were performed, and lignin peroxidase (LiP), laccase (Lac), dye-decolorizing peroxidase (DyP) and aryl-alcohol oxidase (AAO) activities were detected. A 55.3% reduction in the molecular weight (Mw) of KL was observed after 7 days of incubation with Streptomyces sp. S6 based on gel-permeation chromatography (GPC). Gas chromatography-mass spectrometry (GC-MS) also successfully highlighted the production of lignin-derived aromatic compounds, such as 3-methyl-butanoic acid, guaiacol derivatives, and 4,6-dimethyl-dodecane, after treatment of KL with strain S6. Finally, draft genome analysis of Streptomyces sp. S6 also revealed the presence of strong lignin degradation machinery and identified various candidate genes responsible for lignin depolymerization, as well as for the mineralization of the lower molecular weight compounds, confirming the lignin degradation capability of the bacterial strain.


Assuntos
Lignina/metabolismo , Streptomyces/enzimologia , Oxirredutases do Álcool/genética , Biodegradação Ambiental , Biomassa , Etanol/metabolismo , Lacase/genética , Lignina/química , Lignina/genética , Peroxidase/genética , Peroxidases/genética , Polimerização , Streptomyces/genética
20.
Sci Rep ; 10(1): 7942, 2020 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-32409719

RESUMO

In the near future, the demand for L-asparaginase is expected to rise several times due to an increase in its clinical and industrial applications in various industrial sectors, such as food processing. Streptomyces sp. strain NEAE-K is potent L-asparaginase producer, isolated and identified as new subsp. Streptomyces rochei subsp. chromatogenes NEAE-K and the sequence data has been deposited under accession number KJ200343 at the GenBank database. Sixteen different independent factors were examined for their effects on L-asparaginase production by Streptomyces rochei subsp. chromatogenes NEAE-K under solid state fermentation conditions using Plackett-Burman design. pH, dextrose and yeast extract were the most significant factors affecting L-asparaginase production. Thus, using central composite design, the optimum levels of these variables were determined. L-asparaginase purification was carried out by ammonium sulfate followed by DEAE-Sepharose CL-6B ion exchange column with a final purification fold of 16.18. The monomeric molecular weight of the purified L-asparaginase was 64 kD as determined by SDS-PAGE method. The in vitro effects of L-asparaginase were evaluated on five human tumor cell lines and found to have a strong anti-proliferative effects. The results showed that the strongest cytotoxic effect of L-asparaginase was exerted on the HeLa and HepG-2 cell lines (IC50 = 2.16 ± 0.2 and 2.54 ± 0.3 U/mL; respectively). In addition, the selectivity index of L-asparaginase against HeLa and HepG-2 cell lines was 3.94 and 3.35; respectively.


Assuntos
Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Asparaginase/biossíntese , Asparaginase/farmacologia , Streptomyces/metabolismo , Antineoplásicos/isolamento & purificação , Asparaginase/isolamento & purificação , Linhagem Celular Tumoral , Humanos , Filogenia , Streptomyces/enzimologia
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